The field of this invention is analytical instruments, particularly matrix-assisted laser desorption/ionization (xe2x80x9cMALDIxe2x80x9d) instruments.
Matrix-assisted laser desorption/ionization (xe2x80x9cMALDIxe2x80x9d) is a process of ionizing analytes in a sample in a manner that allows the ionized analytes to be further studied. During the past decade, MALDI has proven to be a valuable tool in the analysis of molecules, e.g., biomolecules or biosubstances, and especially large molecules and has application in a wide variety of fields such as genomics, proteomics and the like. Accordingly, a number of MALDI devices have been developed for performing MALDI on an analyte of interest, where in certain instances these MALDI devices are coupled to or otherwise integrated with a device for studying the MALDI ionized analyte, e.g., mass spectrometers. Mass spectrometers are instruments that measure and analyze ions by their mass and charge. For the most part, time-of-flight mass spectrometers (xe2x80x9cTOF-MSxe2x80x9d) are used for this purpose, but other mass spectrometers may be used as well, such as ion cyclotron resonance spectrometers (Fourier transform ion cyclotron mass resonance) and high-frequency quadrupole ion trap mass spectrometers.
Generally, MALDI is a method that enables vaporization and ionization of non-volatile biological analytes from a solid phase directly into a gas phase. To accomplish this task, the analyte of interest is suspended or dissolved in a matrix that generally is a small organic compound that co-crystallizes with the analyte. A sample containing the analyte/matrix mixture is then applied to a suitable support, e.g., a sample probe or sample plate, which is then loaded into device for performing MALDI. It is theorized that the presence of the matrix enables the analyte to be ionized without being degraded, a problem of other analogous methods. Accordingly, MALDI enables the detection of intact molecules as large as 1 million Daltons.
A laser beam serves as the desorption and ionization source in MALDI and, as such, once the substrate supported sample is properly loaded into the MALDI device, a laser is used to vaporize the analyte. In the vaporization process, the matrix absorbs some of the laser light energy causing part of the illuminated matrix to vaporize. The resultant vapor cloud of matrix carries some of the analyte with it so that the analyte may be analyzed. As such, the matrix molecules absorb most of the incident laser energy, thus minimizing analyte damage and ion fragmentation.
Once the molecules of the analyte are vaporized and ionized, they may be analyzed. As mentioned above, this may be accomplished by the use of a mass spectrometer. Accordingly, the vaporized ions are transferred electrostatically into a mass analyzer where they are separated from the matrix ions, for example a TOF-MS flight tube. Following separation of the ions, the ions are then directed to a detector so that the ions may be individually detected. Depending on the nature of the analyzer and how it separates the ions, mass spectrometers fall into different categories. In the case of a TOF-MS for example, separation and detection is based on the mass-to-charge (m/z) ratios of the ions. As such, detection of the ions at the end of the time-of-flight tube is based on their flight time, which is proportional to the square root of their m/z.
When designing effective MALDI methods, attention must be given to the support upon which a sample of the matrix/analyte mixture is applied so that it can be inserted into an appropriate MALDI device. These supports may range from single sample supports to multi-sample supports similar to conventional microtiter plates. Regardless of the number of samples accommodated by the support, the procedure for applying a sample to the support is generally the same. In depositing a sample for analysis onto a sample support, the sample must be deposited at a specific position on the supports where in many embodiments it is dried. This specific position corresponds to the position of the laser beam and also provides a unique address for the sample such that identification of a particular sample, amongst multiple samples analyzed, is possible.
It will be apparent that for MALDI protocols it is important to be able to position the sample at a particular area of the support with a high degree of precision and accuracy so that the sample is not only positioned in the correct position, but also so that there is no cross-contamination between samples if more than one sample is present on a substrate, i.e., the sample is retained at the particular position. Without visual aids, it is difficult, particularly for manually deposited samples, to precisely and accurately position the small volumes of sample required, even with the use of a pipette. Furthermore, even if a sample is precisely positioned on a support, the sample may spread or wick out of the area and could contaminate the other samples, if present, or deplete the amount of sample in the intended area that is to be interrogated by the MALDI laser to a level that may be below the minimum volume requirements for MALDI.
Prior solutions intended to provide discrete positions at which to deposit a sample for MALDI have thus far not provided complete solutions. For example, supports having surfaces with scribed patterns (laser etched, chemically etched, and the like) have been developed. However, while laser scribed surfaces may provide visual clues to a particular location of a support, these laser scribed patterns usually do not effectively contain the sample in the location and thus the sample may still spread about the support surface and in fact may even facilitate wicking the sample out of the designated support location. The problems associated with laser scribed surfaces are only exacerbated by the use of large sample volumes.
Patterning the support surface, e.g., with a hydrophobic/hydrophilic treatment or the like, has also been attempted. These patterns, such as hydrophobic/hydrophilic patterns, are surface treatments that are typically a film or a chemically modified monolayer on the support surface. While these patterns may contain a sample to a specific area of the support once the sample is deposited thereto, they are difficult, if not impossible, to see with the naked human eye and thus usually do not provide a visual reference to aid in depositing a sample at a particular support location. Furthermore, these patterned areas usually have a sample volume limit such that once this limit is exceed, the sample spreads out of the designated area thus depleting the sample volume for analysis and/or contaminating other samples, if present.
As such, there continues to be an interest in the development of supports or sample holders suitable for use in MALDI protocols. Of particular interest are supports that provide visual references or guides to designated areas on the support, effectively contain a sample in a designated area, are cost effective and easy to manufacture, arc able to accommodate a wide range of sample volumes, do not adversely affect the desorption/ionization of the sample, and which may be provided in a wide variety of configurations including single sample supports, as well as multiple sample supports that are able to accommodate a plurality of samples without cross-contamination.
References of interest include: International Publication Nos.: WO 99/63576; GB 2,312782 A; GB 2,332,273 A; GB 2,370114A; and EP 0964427 A2, as well as in U.S. Patent Publication No. 2002031773; and U.S. Pat. Nos.: 5,498,545; 5,643,800; 5,777,324; 5,777,860; 5,828,063; 5,841,136; 6,111,251; 6,287,872; 6,414,306; and 6,423,966; the disclosures of which are herein incorporated by reference.
MALDI sample holders and methods of using and making the same are provided. The MALDI sample holders are configured for use in matrix assisted laser desorption/ionization and include a planar substrate having a surface and at least one fluid retaining structure present on the surface. The fluid retaining structure includes a material that changes from a first fluid state to a second solid state in response to a stimulus. Also provided arc methods of using the subject MALDI sample holders in a matrix-assisted laser desorption/ionization protocol that include providing a subject MALDI sample holder, depositing a sample into at least one fluid retaining structure of the MALDI sample holder, inserting the MALDI sample holder into a matrix assisted laser desorption/ionization device and performing matrix assisted laser desorption/ionization. The subject invention also includes methods of producing the subject MALDI sample holders that include providing a planar substrate having a surface and providing a material in first fluid state. In the subject methods, the material is positioned on the substrate surface and a stimulus is applied to the material to change it to a second solid state. The stimulus may be applied either before or after the material is positioned on the substrate surface. Also provided are kits for use in practicing the subject methods.